KR102402970B1 - PIC-containing composition for the treatment of cancer - Google Patents

Abstract

The present disclosure relates to a composition comprising a PIC for the treatment of cancer. More specifically, the present disclosure relates to a composition for the treatment of cancer comprising polyinosinic acid-polycytidylic acid, an antibiotic or polyamine compound, a positive ion, and optionally a virus, and the use of the composition in the manufacture of a medicament for the treatment of cancer to start There are no publicly available drawings.

Description

PIC-containing composition for the treatment of cancer

This application relates to the medical field, in particular to the treatment of cancer. More specifically, the present application relates to a pharmaceutical composition for the treatment of cancer comprising polyinosinic acid-polycytidylic acid (PIC), an antibiotic or polyamine compound, a positive ion, and optionally an inactivated or attenuated virus; and to its use for the treatment of cancer.

Cancer is often referred to as a malignant tumor. Under the influence of carcinogens, some cells of the tissue lose their normal growth control at the gene level, leading to abnormal proliferation and differentiation, ultimately becoming tumors. Compared to benign tumors, malignant tumors grow faster, are invasive, prone to bleeding, necrosis, ulceration, and often metastasize.

There are over 100 different types of cancers that affect humans. The top five common cancer sites are lung, prostate, colorectal, stomach and liver cancers in men and breast, colorectal, lung, cervical and gastric cancers in women. Jie He and Wanqing Chen et al. published a paper titled Cancer Statistics in China in 2015 in the Cancer Journal for Clinicians. This study analyzed and predicted the incidence and mortality rates of malignant tumors in China in 2015. The data indicated that 4,292,000 new malignant tumor cases were expected in 2015 in China. The five most common cancers are lung and bronchial cancer, stomach cancer, liver cancer, esophageal cancer and colorectal cancer in men and breast cancer, lung cancer, stomach cancer, colorectal cancer and esophageal cancer in women. Lung cancer is one of the most common malignant tumors in China and has the highest mortality rate from malignant tumors.

According to the World Health Organization (WHO), each year there are approximately 14 million new cancer cases and 8.2 million deaths from cancer, accounting for 13% of all deaths worldwide. Cancer is the leading cause of death in both developed and developing countries. New cancer cases are projected to increase by about 70% over the next 20 years, increasing the global burden (WHO). Therefore, the trend of increasing cancer incidence has pressured mankind to focus more on cancer prevention and treatment.

Recently, primary methods for treating cancer include surgical treatment, chemotherapy using drugs to kill cancer cells, radiation therapy using high-energy radiation to kill cancer cells, and specific cancer cells to deliver targeted therapy. targeted therapy, which uses substances that target, and immunotherapy, which uses the immune system to treat cancer.

In addition to this, the application of viruses in cancer treatment is attracting increasing attention. In 1912, DePace discovered cervical tumor regression after rabies vaccine was administered to a female patient bitten by a dog. Since then, there have been several reports of the use of the virus to treat cancer. Especially between 1950 and 1960, viral treatment of cancer developed rapidly. In the 1970s, the development of viral therapy was slowed by problems of viral pathogenicity, but its pace recovered again in the 1980s.

Guoqian Kuang et al. described potential mechanisms of anticancer viruses, as well as selection and methods in clinical applications (Current Status and Prospect of Cancer Virotherapy Clinical Studies, Journal of Guangxi Medical University, 1995 Vol. 12: 617-619). Currently, it is recognized that the mechanisms of anticancer viruses include direct oncolytic action, enhancement of the immune system and stimulation of cytokine release. It is generally believed that viruses used to treat cancer should be non-carcinogenic and have good antigenicity. Viruses reported for anticancer therapy include: NDV, mumps virus, vaccinia virus, Sendai virus, HSV and parvovirus (Kenney S et al., Viruses as oncolytic agents: a new age for "therapeutic") viruses.J Nati Cancer Inst, 1994,86: 1 185). Lorence et al. found that live virus showed significantly better anticancer effect in animal study compared to inactivated virus (Complete regression of human neuroblastoma xenografts in athymic mice after local Newcastle disease virus therapy. J Nati Cancer Inst, 1994, 86 :1228).

Some viruses specifically kill tumor cells in cancer treatment, and they are known as oncolytic viruses. Oncolytic viruses can selectively replicate inside cancer cells, resulting in cytopathic effects and immune responses leading to tumor cell death, while showing minimal effects on normal cells and tissues (Jiang Zhong, Oncolytic Virus and Tumor Treatment, Foreign Medicine (Microbiology Section), 2004 Vol. 27 Iss. No. 6).

Currently, there are many reports of oncolytic viruses in cancer treatment. For example, WO2009/016433 discloses a recombinant non-VSV Radovirus (eg Maraba virus, Carajas virus, Muir Springs virus) to treat hyperproliferative diseases (eg cancer). ), and/or Bahia grande virus). US2010/0297072A1 is an oncolytic virus (paramyxovirus, reovirus, herpesvirus, adenovirus, and Semliki Forest virus) and an immunostimulant (CTLA-) 4 A composition for treating cancer comprising a blocker, IL-21, anti-CD40 or granulocyte-macrophage colony stimulating factor (GM-CSF)), and administering the composition to MAC205 fibrosarcoma or B16 melanoma tumor-bearing mice A significant inhibition of tumor growth is disclosed.

Rabies virus belongs to the genus lyssavirus of the family rhabdoviridae. The rabies virus has a distinct "bullet" shape, has a helical, symmetrical nucleocapsid with a shell, and contains a single-stranded RNA interior. Rabies virus is the pathogen for rabies disease. Anti-rabies immunization is one of the successful early vaccination practices. In the 1880s, Pasteur pioneered the use of vaccines. Early rabies vaccines were neural tissue-derived, and embryonic egg-culture vaccines, cell culture vaccines, and subunit vaccines were subsequently developed. Genetically pioneered vaccines are under development (Shounan Tan, Fengyu Zhang, Studies on Rabies and Human Rabies Vaccine. Medical Information 201 1 Vol. 24:2841 -2842). Currently, most of the rabies vaccines for use in humans are obtained by inoculating Vero cells with a fixed virus strain (eg, CTN-1V strain, aG strain), culturing the virus to obtain a liquid, followed by inactivation, concentration and purification. It is produced by freeze-drying, followed by further addition of an appropriate amount of gelatin or sucrose as a protective agent (Yuhui Zhang, The Establishment of Rabies Vaccine Purification Technology. Chinese Journal of Biologicals 1999 Vol. 12 Iss. No. 4: 231 - 232).

Jieguang Sun et al. reported that the side effects of malignant tumor treatment were improved in an animal tumor model by daily use of a rabies vaccine for human use as the sole active ingredient via an intramuscular injection route at a dose of 2.5 IU to 10 IU, which also was found to enhance phagocytosis (see CN100341571 C). RU2414238C2 also reports how the rabies vaccine increases cancer resistance, increasing the antitumor resistance of the organism to 9,10-dimethyl-1,2-benzanthracene.

Viruses have great potential for cancer treatment. Safer and more effective virus-based anticancer drugs are needed in the field.

The present disclosure provides for use in the treatment of cancer comprising or consisting of a) polyinosinic acid-polycytidyl acid (PIC), b) at least one antibiotic or at least one polyamine compound, and c) at least one positive ion. It provides a composition for

In another aspect, the present disclosure provides for a) a) polyriboinosinic acid-polyribocytidylic acid (PIC), b) at least one antibiotic or at least one polyamine compound, c) at least one positive ion, and d) optionally a virus. Provided is a composition for use in the treatment of cancer, comprising or consisting thereof.

In one embodiment, the composition comprises a) polyribinosinic acid-polyribocytidylic acid (PIC), b) at least one antibiotic or at least one polyamine compound, c) at least one positive ion, and d) a virus. .

In some embodiments, the virus is inactivated, attenuated, or unable to replicate in a human subject. In some specific embodiments, the virus is inactivated.

In some embodiments, the virus is rhabdoviridae , adeniviridae , arenaviridae , astroviridae , bunyaviridae , cliciviridae , flaviviridae ( flaviviridae ), hepatitis delta virus , hepeviridae , mononegavi rales , nidovirales , piconaviridae , orthomyxoviridae selected from the group consisting of papillomaviridae , parvoviridae , polyomaviridae , poxviridae , reoviridae , retroviridae , and togaviridae do. In some specific embodiments, the virus belongs to the genus Lysavirus of the Ravdovirus family. In some specific embodiments, the virus is a rabies virus.

In some embodiments, the rabies virus is an inactivated crude virus (such as hamster kidney cell inactivated crude rabies antigen (HKC-ICRA)), or an inactivated purified virus (such as hamster kidney cell inactivated purified rabies antigen (HKC-IPRA). ))to be.

In some embodiments, rabies viruses suitable for use in the present disclosure are rabies vaccines, including but not limited to inactivated, subunit, genetically modified, and polypeptide vaccines. In particular, suitable rabies virus vaccines for use in the present disclosure include human diploid cell vaccine (HDCV), or hamster kidney cell inactivated purified rabies vaccine (HKC-IPRV), or hamster kidney cell inactivated crude rabies vaccine (HKC- ICRV), or purified Vero cell rabies vaccine (PVRV), or purified chicken embryonic cell (PCEC) rabies vaccine, or purified duck embryonic rabies vaccine (PDEV).

In some embodiments, the PIC is heterogeneous with respect to molecular weight, wherein the molecular weight is at least 66,000 Daltons. A value of 66,000 Daltons corresponds to a molecular size of 6.4 sedimentation units (Svedbergs). In some embodiments, the molecular weight of the PIC is 66,000 to 1,200,000 Daltons (equivalent to 6.4 to 24.0 sedimentation units). In some other embodiments, the molecular weight of the PIC is at least 150,000 Daltons. In some other embodiments, the molecular weight of the PIC is between 100,000 and 200,000 Daltons, or between 300,000 and 4,000,000 Daltons, or between 500,000 and 1,000,000 Daltons, or between 1,000,000 and 1,500,000 Daltons, or between 1,500,000 and 2,000,000 Daltons, or between 2,000,000 and 2,500,000 Daltons, or between 2,500,000 and 2,500,000 Daltons. 3,000,000 Daltons, or 3,000,000 to 3,500,000 Daltons, or 3,500,000 to 4,000,000 Daltons, or 4,000,000 to 4,500,000 Daltons, or 4,500,000 to 5,000,000 Daltons.

In some specific embodiments, the antibiotic is tacrolamycin, anthracycline, butyrin sulphate, gentamicin, hygromycin, amikacin, dideoxy kanamycin, nebramycin, β-lactam, neomycin, puromycin, streptomycin, streptozocin, and any combination thereof selected from the group consisting of Polyamine compounds include arginine salt, spermidine, N-(3-aminopropyl), N-(3-aminopropyl)-1,4-butanediamine, spermine, OS-dimethylaminothiophosphate, poly-lysine, amino glycosides, and any combination thereof.

In some specific embodiments, the antibiotic is kanamycin. In some embodiments, the concentration of the antibiotic in the composition is between 10 Units/ml and 100,000 Units/ml, preferably between 100 Units/ml and 10,000 Units/ml, more preferably between 500 Units/ml and 5,000 Units/ml.

In some embodiments, the positive ion is selected from the group consisting of calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, zinc, and any combination thereof. In some specific embodiments, the positive ion is calcium. The positive ion may be in the form of any suitable salt or organic complex, including but not limited to chloride, fluoride, hydroxide, phosphate or sulfate. For example, when the positive ion is calcium, the calcium ion is calcium carbonate , calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate. In some embodiments, the concentration of positive ions in the composition is 0.01 μmol to 10 mmol/ml, preferably 0.02 μmol to 5 mmol/ml, more preferably 0.1 μmol to 1 mmol/ml, most preferably O .1 μmol to 100 μmol/ml.

In some embodiments, the ratio of virus to PIC is 1 IU/50 μg, 1 IU/60 μg, 1 IU/70 μg, 1 IU/80 μg, 1 IU/90 μg, 1 IU/100 μg, 1 IU/ 125 μg, 1 IU/200 μg, 1 IU/250 μg, 1 IU/300 μg, 1 IU/350 μg, 1 IU/400 μg, 1 IU/450 μg, 1 IU/500 μg, 1 IU/550 μg, 1 IU/600 μg, 1 IU/700 μg, 1 IU/800 μg, 1 IU/1OOO μg, 1 IU/1500 μg, 1 IU/2000 μg, 1 IU/2500 μg, 1 IU/3000 μg, 1 IU/4000 μg, 1 IU/5000 μg, 1 IU/6000 μg, 1 IU/7000 μg, 1 IU/8000 μg, 1 IU/9000 μg, 1 IU/10000 μg, and ranges between any two of the above ratios is selected from the group being In particular, the ratio of the virus to the PIC is 1 IU/500 μg.

In some embodiments, the amount of PIC in the composition is between 250 μg and 5000 μg per unit dose; For example, the amount of PIC is selected from the group consisting of 250 μg, 500 μg, 1000 μg, 1500 μg, 2000 μg, 3000 μg, 4000 μg, 5000 μg per unit dose and a range between any two of the above amounts. do.

In some specific embodiments, the amount of PIC in the composition is between 500 μg and 4000 μg per unit dose, or between 1000 μg and 3000 μg per unit dose, or between 1000 μg and 2500 μg per unit dose.

When the composition of the present disclosure is applied to an adult, the amount of PIC in the composition is selected from the group consisting of 500 μg, 1000 μg, 1500 μg, 2000 μg per unit dose and a range between any two of the above amounts. When the composition of the present disclosure is applied to a minority (eg, children), the amount of PIC in the composition is selected from the group consisting of 250 μg, 500 μg, 1000 μg, 1250 μg per unit dose and a range between any two of the above amounts. do.

In some embodiments, a unit dose of the present disclosure is 0.1 ml, 0.15 ml, 0.2 ml, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, 4.0 ml, 5.0 ml, 10.0 ml, 20.0 ml, from the group consisting of 30.0 ml, 40.0 ml, 50.0 ml, 60.0 ml, 70.0 ml, 80.0 ml, 90.0 ml, 100.0 ml, 150.0 ml, 200.0 ml, 250.0 ml, and a range between any two of the above volumes. prepared in a selected volume. It will be appreciated by those skilled in the art that administration volumes that are too large or too small cause discomfort in clinical practice. Therefore, when the composition of the present disclosure is administered to a human subject, the unit dose is preferably in the range of 0.5 ml to 1.0 ml for injection, preferably in the range of 0.15 ml to 0.2 ml for intranasal administration, preferably For intravenous injection it ranges from 30.0 ml to 100.0 ml. Although unit doses are expressed in terms of volume, it is recognized that these doses do not imply that the compositions of the present disclosure may only be in liquid dosage form. When the composition of the present disclosure is prepared in a solid dosage form (dry or lyophilized powder), the volume per unit dose refers to the volume of solution after reconstitution of the dried or lyophilized powder.

In some embodiments, the amount of virus in the composition is between 0.1 IU and 100.0 IU per unit dose or between 0.2 and 100.0 IU per unit dose. In particular, the amount of virus in the composition is 0.2 IU, 0.5 IU, 1.0 IU, 1.5 IU, 2.0 IU, 2.5 IU, 3.0 IU, 3.5 IU, 4.0 IU, 5.0 IU, 6.0 IU, 7.0 IU, 8.0 IU, 9.0 per unit dose. IU, 10.0 IU, 15.0 IU, 20.0 IU, 30.0 IU, 40.0 IU, 50.0 IU, 60.0 IU, 70.0 IU, 80.0 IU, 90.0 IU, 100.0 IU, and a range between any two of the above amounts. is selected from In some specific embodiments, the amount of virus in the composition is 0.5 IU to 3.0 IU per unit dose; Preferably it is 1.0 IU to 2.5 IU per unit dose. In some specific embodiments, when applied to adults, the amount of virus in the composition is between 0.5 IU and 10.0 IU per unit dose. In some other specific embodiments, when applied to a minority, the amount of virus in the composition is between 0.5 IU and 5.0 IU per unit dose.

In some embodiments, the concentration of virus in the composition is between 0.05 IU/ml and 40.0 IU/ml; Preferably 0.05 IU/ml, 0.1 IU/ml, 0.15 IU/ml, 0.2 IU/ml, 0.5 IU/ml, 1.0 IU/ml, 2.0 IU/ml, 3.0 IU/ml, 4.0 IU/ml, 5.0 IU /ml, 10 IU/ml, 15 IU/ml, 20 IU/ml, 25 IU/ml, 30 IU/ml, 35 IU/ml, 40 IU/ml.

In some embodiments, the composition of the present disclosure further comprises at least one auxiliary substance as an excipient and a stabilizer. Auxiliary substances are gelatin, sucrose, sugar, lactose, maltose, trehalose, glucose, low molecular weight dextran, sorbitol, polysorbate 20, mannitol polyethylene glycol, human serum albumin, recombinant albumin, sodium octanoate, urea, hydroxide aluminum, phenol red, magnesium chloride, potassium chloride, sodium chloride, sodium thiosulfate, potassium dihydrogen phosphate, ascorbic acid, trichloromethane, phenol, and thimerosal and any combinations thereof.

In some embodiments, the compositions of the present disclosure comprise at least one physiologically acceptable buffer selected from the group consisting of acetate, tris(hydroxymethyl)aminomethane (tris), bicarbonate, carbonate, phosphate buffers, and any combination thereof. further includes. The pH of the buffer or composition is 6.50, 6.60, 6.70, 6.80, 6.90, 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45, 7.50, 7.55, 7.60, 7.65, 7.70, 7.75, 7.80, 7.85, 7.90, 7.95, 8.00, and a range between any two of the above pH values. In one embodiment, the pH of the composition is between pH 6.5 and pH 8.0. In some specific embodiments, the buffer is PBS and the pH of the buffer is between 7.3 and 7.5.

Compositions of the present disclosure may be prepared in solid dosage forms or liquid dosage forms. Compositions of the present disclosure may be prepared in a dry powder, liquid solution or liquid dosage form (such as an injectable solution, aqueous or physiological saline solution, suspension, ointment, drop, emulsion, gel, syrup or serofluid), tablet, coated tablet, It may be prepared in a form selected from the group consisting of microcapsules, suppositories, granules, dragees, and capsules. Methods of preparation are generally described in Vaccine 4th Edition (Stanley A Plotkin et al., W. B. Saunders Company 2003). Preferably, the composition of the present disclosure is prepared in the form of an injectable solution.

In another aspect, the present disclosure provides a composition for use in the treatment of cancer, in the form of a dry powder or lyophilized powder.

In another aspect, the present disclosure provides use of a combination of a PIC, at least one antibiotic (or at least one polyamine compound) and at least one positive ion, in the manufacture of a medicament for the treatment of cancer.

In another aspect, the present disclosure provides the use of a combination comprising a PIC adjuvant disclosed in CN103405762A in the manufacture of a medicament for the treatment of cancer.

In another aspect, the present disclosure provides use of a combination of a PIC, at least one antibiotic (or at least one polyamine compound), at least one positive ion and a virus, in the manufacture of a medicament for the treatment of cancer.

In another aspect, the present disclosure provides use of a composition for the treatment of cancer.

More specifically, the present disclosure provides for the use of a composition in the manufacture of a medicament for the treatment of cancer.

In some specific embodiments, the cancer is selected from the group consisting of:

Oropharyngeal cancer, nasopharyngeal carcinoma, esophageal cancer, stomach cancer, colon cancer, liver cancer, cholangiocarcinoma, bladder cancer, pancreatic cancer, lung cancer, tracheal cancer, thymoma, bone cancer, junction cancer, melanoma, mesothelial cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer, brain cancer, myeloma, blood cancer;

Malignant neoplasm of the lips, malignant neoplasm of the root of the tongue, malignant neoplasm of the gums, malignant neoplasm of the oral cavity, malignant neoplasm of the palate, malignant neoplasm of the parotid gland, malignant neoplasm of the tonsils, malignant neoplasm of the oropharynx, malignant neoplasm of the nasopharynx organism, malignant neoplasm of piriform sinus, malignant neoplasm of hypopharynx;

malignant neoplasm of esophagus, malignant neoplasm of stomach, malignant neoplasm of small intestine, malignant neoplasm of colon, malignant neoplasm of rectosigmoid junction, malignant neoplasm of rectum, malignant neoplasm of anus and anal canal , malignant neoplasms of the liver and intrahepatic bile ducts, malignant neoplasms of the gallbladder, malignant neoplasms of the pancreas;

Nasal and middle ear malignant neoplasms, sinus malignant neoplasms, laryngeal malignant neoplasms, tracheal malignant neoplasms, bronchial and lung malignant neoplasms, thymic malignant neoplasms, cardiac, mediastinal and pleural malignancies neoplasm;

bone and articular cartilage malignant neoplasms;

cutaneous malignant melanoma;

mesothelial and soft tissue malignant neoplasms;

breast malignant neoplasm;

genital malignant neoplasm, vaginal malignant neoplasm, cervical malignant neoplasm, uterine malignant neoplasm, ovarian malignant neoplasm, placental malignant neoplasm;

penile malignant neoplasm, prostate malignant neoplasm, testicular malignant neoplasm;

malignant neoplasms of the urinary tract;

malignant neoplasms of the eye and appendages;

malignant neoplasms of the meninges, brain malignant neoplasms;

spinal cord, cranial nerve and central nervous system malignant neoplasms;

endocrine malignant neoplasm;

Hodgkin's disease, non-Hodgkin's lymphoma of follicular nodule, diffuse non-Hodgkin's lymphoma, peripheral and cutaneous T-cell lymphoma, multiple myeloma, malignant plasma cell tumor, lymphocytic leukemia, myeloid leukemia, monocytic leukemia.

In some specific preferred embodiments, the composition of the present disclosure is for the treatment of a cancer selected from the group consisting of lung cancer, breast cancer, thyroid cancer, kidney cancer, gastric adenocarcinoma, liver cancer, melanoma, tongue cancer, rectal cancer, endometrial cancer, and ovarian cancer is used for

In a further preferred embodiment, the compositions of the present disclosure are used for the treatment of metastatic tumors.

In a further embodiment, the present disclosure provides for use of a composition in combination with an anti-tumor treatment regimen in the manufacture of a medicament for the treatment of cancer;

Preferably, said anti-tumor treatment regimen is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, and immunotherapy, wherein the therapeutic agent used in said chemotherapy is an alkylating agent, an anti-metabolic anti-neoplastic agent, an anti- selected from the group consisting of tumor antibiotics, anti-tumor plants, platinum compound anti-neoplastic agents, hormone balancing anti-neoplastic agents, and several kinds of anti-neoplastic agents, wherein the therapeutic agent used in the targeted therapy is rituximab; Group consisting of bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, and sorafenib (sorafenib) wherein the therapeutic agent used in the immunotherapy is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor and a CTLA4 inhibitor; More preferably, the alkylating agent is selected from the group consisting of cyclophosphamide, ifosfamide and thiotepa, and the anti-metabolic antineoplastic agent is composed of methotrexate, mercaptopurine, fluorouracil and cytarabine. wherein said anti-tumor antibiotic is selected from the group consisting of bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin and mitoxantrone, wherein said anti-tumor plant is selected from the group consisting of vincristine, etoposide, teniposide, paclitaxel and docetaxel, the platinum compound anti-neoplastic agent is selected from the group consisting of cisplatin, carboplatin and oxaliplatin, and the hormone balancing anti-neoplastic agent leuprol It is selected from the group consisting of lead, tamoxifen, flutamide and formestane, and the various anti-neoplastic agents are arsenic trioxide.

Alternatively, the present invention encompasses the use of a composition disclosed herein in the manufacture of a medicament for the treatment of cancer; wherein the medicament is for use in combination with an anti-tumor treatment regimen. Preferably, said anti-tumor treatment regimen is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, and immunotherapy, wherein the therapeutic agent used in said chemotherapy is an alkylating agent, an anti-metabolic anti-neoplastic agent, an anti- selected from the group consisting of tumor antibiotics, anti-tumor plants, platinum compound anti-neoplastic agents, hormone balancing anti-neoplastic agents, and several kinds of anti-neoplastic agents, wherein the therapeutic agent used in the targeted therapy is rituximab, bevacizumab , trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, and sorafenib, wherein the therapeutic agent used in the immunotherapy is a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA4 inhibitor. selected from the group consisting of; More preferably, the alkylating agent is selected from the group consisting of cyclophosphamide, ifosfamide and thiotepa, and the anti-metabolic antineoplastic agent is composed of methotrexate, mercaptopurine, fluorouracil and cytarabine. wherein said anti-tumor antibiotic is selected from the group consisting of bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin and mitoxantrone, wherein said anti-tumor plant is selected from the group consisting of vincristine, etoposide, teniposide, paclitaxel and docetaxel, the platinum compound anti-neoplastic agent is selected from the group consisting of cisplatin, carboplatin and oxaliplatin, and the hormone balancing anti-neoplastic agent leuprol It is selected from the group consisting of lead, tamoxifen, flutamide and formestane, and the various anti-neoplastic agents are arsenic trioxide.

The invention also encompasses the use of a composition disclosed herein in the manufacture of a medicament for the treatment of cancer and the use of at least one additional anti-neoplastic agent in the manufacture of a medicament for the treatment of cancer. The invention also encompasses the use of the compositions disclosed herein and at least one additional anti-neoplastic agent in the manufacture of a medicament for the treatment of cancer.

Preferably, the anti-tumor compound is selected from the group consisting of a chemotherapeutic agent, a targeted therapeutic agent and an immunotherapeutic agent.

Preferably, the chemotherapeutic agent is selected from the group consisting of alkylating agents, anti-metabolic anti-neoplastic agents, anti-tumor antibiotics, anti-tumor plants, platinum compound anti-neoplastic agents, hormone balancing anti-neoplastic agents, and several types of anti-neoplastic agents. do.

Preferably, the targeted therapeutic is selected from the group consisting of rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, and sorafenib.

Preferably, the immunotherapeutic agent is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor and a CTLA4 inhibitor.

Preferably, the alkylating agent is selected from the group consisting of cyclophosphamide, ifosfamide and thiotepa.

Preferably, the anti-metabolic antineoplastic agent is selected from the group consisting of methotrexate, mercaptopurine, fluorouracil and cytarabine.

Preferably, the anti-tumor antibiotic is selected from the group consisting of bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin and mitoxantrone.

Preferably, said anti-tumor plant is selected from the group consisting of vincristine, etoposide, teniposide, paclitaxel and docetaxel.

Preferably, the platinum compound anti-neoplastic agent is selected from the group consisting of cisplatin, carboplatin and oxaliplatin.

Preferably the hormonal anti-neoplastic agent is selected from the group consisting of leuprolide, tamoxifen, flutamide and formestane.

Preferably, the various anti-neoplastic agents are arsenic trioxide.

In the context of the present disclosure, when a composition of the present disclosure comprises a virus, the cancer to be treated is not caused by the virus in the composition. In some specific embodiments, the cancer being treated is not caused by the rabies virus.

In another aspect, the present disclosure provides a method of treating cancer comprising administering to a subject, eg, a human subject, a therapeutically effective amount of the composition.

In some embodiments, administration of the composition is systemic or localized. In some embodiments, the composition is administered via parenteral injection (eg, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, intratumoral, peritumoral). In some other embodiments, the composition is administered intradermally via a route other than injection (eg, a route that does not disrupt the endothelial cell barrier by mechanical means). In some other embodiments, the composition is administered via the rectal, vaginal, nasal (eg intranasal), oral, buccal, sublingual, respiratory, ocular (eg intraocular), or transdermal route.

In particular, examples of routes of administration include intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, intradermal, intranasal, intraocular, oral, sublingual, intratumoral, and peritumoral.

In some embodiments, a composition of the present disclosure is administered once a month, twice a month, 3 times a month, 4 times a month, 5 times a month, 6 times a month, 7 times a month, 8 times a month, once a week, twice a week, 3 times a week, 4 times a week, 5 times a week, 6 times a week, once every 3 days, 2 times every 3 days, 3 times every 3 days, once every 2 days, twice every 2 days, 1 day a day administered to the human subject based on a frequency selected from the group consisting of twice a day.

In particular, the method comprises administering to a human subject a therapeutically effective amount of a composition according to the invention via intramuscular injection at a frequency of twice every three days, or administering a therapeutically effective amount of a composition according to the invention to a human subject at a frequency of once a week. and administering via intramuscular injection.

In another aspect, the present disclosure provides a pharmaceutical kit or kit for use in practicing the method of treatment comprising at least one container, each of which independently comprises a composition of the present disclosure. The composition and/or amount of the composition in the different containers may be the same or different.

In some embodiments, kits of the present disclosure comprise at least one container containing 2.0 IU/ml of inactivated rabies virus and 1000 pg/ml of PIC.

In some embodiments, the compositions of the present disclosure are formulated as sterile liquids and contained in sterile containers (eg, tubes, bottles, ampoules, syringes). In another embodiment, a composition of the present disclosure is contained in a container in the form of a dry or lyophilized powder that is restored to a liquid form prior to use.

In some embodiments, the kit of the present disclosure further comprises an item selected from the group consisting of a needle, water for injection, instructions for use, and any combination thereof.

Justice

As used herein, the term "comprising" is construed as specifying the presence of the stated feature, integer, step or component being referred to, but of one or more features, integers, steps or components, or groups thereof. The presence or addition is not excluded. However, in the context of this disclosure, the term “comprising” also includes “consisting of”. Variations of the word "comprising", such as "comprises" and "comprising" and "comprising", have correspondingly changed meanings.

"Inactivated" indicates that the viral pathogenicity and/or replication ability is eliminated, but retains the ability to induce a protective immune response in the human body. Virus inactivation strategies are well known in the art. Any conventional method may be used to inactivate a virus, and a suitable method may be selected for a specific virus type. Methods of virus inactivation include, but are not limited to: photoreactive compounds, oxidizing agents, radiation (such as UV radiation, γ-radiation), UV radiation in combination with riboflavin, solvent/surfactant (S/D) treatment (such as tri (n-butyl)phosphate and/or using Tween 80), polyethylene glycol (PEG) treatment, pasteurization (heat treatment), acidic pH treatment, enzyme treatment (pepsin or trypsin), dimethyl methylene blue and visible light Using methylene blue light treatment, psoralen derivative S-59 and UVA irradiation treatment.

In "attenuated", the attenuated virus is still viable but its virulence has been reduced during the manufacturing process. Attenuated viruses retain the ability to replicate and stimulate the body to reduce the immune response.

The term “heterogeneity” as used herein in the context of a composition of the present disclosure indicates that the PIC molecules of the composition are not homogeneous with respect to molecular weight, size, or both.

As used herein, the term “unit dose” refers to physically discrete units suitable as a consistent dosage for a human subject. Each unit contains a predetermined amount of the composition sufficient to produce the desired effect in association with a pharmaceutically/physiologically acceptable diluent or carrier.

The terms “treatment,” “treating,” “treat,” and the like are generally used herein to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptoms thereof and/or therapeutic in terms of partial or complete stabilization or cure of the disease and/or side effects that may contribute to the disease. "Treatment" as used herein includes any treatment of a disease in a subject, particularly a human, and includes (i) preventing the disease from developing in a subject who may be afflicted with the disease but has not yet been diagnosed as having the disease; (ii) inhibiting the disease, ie, arresting the development of the disease; or (iii) alleviating the disease, ie causing regression of the disease.

In this disclosure, when describing a numerical range, the expressions "... to...", "within a range", "range between", and the like include endpoint values.

The term virus as used herein refers to a whole virus or a fragment of a virus and includes viral antigens as well as viral vaccines.

The following description of the examples and drawings and drawings is for the purpose of explicit illustration of the invention only and is not intended to limit the scope of the invention.

1 shows the structures of PIC, antibiotics (or polyamine compounds) and positive ions observed under electron microscopy.
2A depicts the tumor volume of LL/2 tumor-bearing mice after treatment. ■ Vehicle control group, ● Cisplatin group, △ YS-ON-001 low-dose group, ◇ YS-ON-001 high-dose group. Bars represent SEM. **: p<0.01 vs. vehicle control.
2B depicts the relative tumor proliferation rates (T/C) of LL/2 tumor-bearing mice after treatment. ● Cisplatin group, △ YS-ON-001 low-dose group, ◇ YS-ON-001 high-dose group.
3 depicts tumor weights of LL/2 tumor-bearing mice after treatment. **: p<0.01 vs. vehicle control. #: p<0.05 vs. YS-ON-001 high capacity group.
4 depicts body weights of LL/2 tumor-bearing mice after treatment. ■ Vehicle control group, ● Cisplatin group, △ YS-ON-001 low-dose group, ◇ YS-ON-001 high-dose group. Bars represent SEM. **: p<0.01 vs. vehicle control.
Figure 5 shows the change in body weight of LL/2 tumor-bearing mice after treatment. ■ Vehicle control group, ● Cisplatin group, △ YS-ON-001 low-dose group, ◇ YS-ON-001 high-dose group. Bars represent SEM. *: p<0.05 vs. vehicle control. **: p<0.01 vs. vehicle control.
6A and 6B depict thymic weight and thymic index of LL/2 tumor-bearing mice after treatment. **: p<0.01 vs. vehicle control.
7A and 7B show spleen weight and spleen index of LL/2 tumor-bearing mice after treatment. *: p<0.05 vs. vehicle control.
8 depicts the tumor volume of 4T1 tumor-bearing mice after treatment. ■ vehicle control group, ● docetaxel group, △ YS-ON-001 low-dose group, ◇ YS-ON-001 high-dose group. Bars represent SEM. **: p<0.01 vs. vehicle control. #: p<0.05 vs. YS-ON-001 high capacity group. ##: p<0.01 vs. YS-ON-001 high capacity group.
9 depicts the relative tumor proliferation rates (T/C) of 4T1 tumor-bearing mice after treatment. ● Docetaxel group, △ YS-ON-001 low-dose group, ◇ YS-ON-001 high-dose group.
10 depicts tumor weights of 4T1 tumor-bearing mice after treatment. Bars represent SEM. **: pO.01 vs. vehicle control. #: p<0.05 vs. YS-ON-001 high capacity group.
11 depicts body weight of 4T1 tumor-bearing mice after treatment. ■ vehicle control group, ● docetaxel group, △ YS-ON-001 low-dose group, ◇ YS-ON-001 high-dose group. Bars represent SEM. *: p<0.05 vs. vehicle control. **: p<0.01 vs. vehicle control.
12 depicts body weight changes of 4T1 tumor-bearing mice after treatment. ■ vehicle control group, ● docetaxel group, △ YS-ON-001 low-dose group, ◇ YS-ON-001 high-dose group. Bars represent SEM. *: p<0.05 vs. vehicle control. **: p<0.01 vs. vehicle control.
13A and 13B depict thymic weight and thymic index of 4T1 tumor-bearing mice after treatment. *: p<0.05 vs. vehicle control. **: p<0.01 vs. vehicle control.
14A and 14B depict spleen weight and spleen index of 4T1 tumor-bearing mice after treatment. *: p<0.05 vs. vehicle control. **: p<0.01 vs. vehicle control.
15A depicts the tumor volume of H22 tumor-bearing mice on treatment. Bars represent SEM. *: p<0.05 vs. vehicle control. **: p<0.01 vs. vehicle control. #: pO.05 vs. Sorafenib+YS-ON-001 group p. ##: p<0.01 vs. Sorafenib+YS-ON-001 group.
15B depicts the relative tumor proliferation rates (T/C) of H22 tumor-bearing mice on treatment.
16A depicts tumor volumes of LL/2 tumor-bearing mice after treatment. Bars represent SEM. **: p<0.01 vs. vehicle control.
16B depicts the relative tumor growth rates (T/C) of LL/2 tumor-bearing mice after treatment.
17 depicts tumor weights of LL/2 tumor-bearing mice after treatment. Bars represent SEM. **: p<0.01 vs. vehicle control.
18A and 18B depict body weights of B16F10 tumor-bearing mice after treatment. Bars represent SEM. *: p<0.05 vs. vehicle control. **: p<0.01 vs. vehicle control.
19 depicts the number of lung metastases in B16F10 tumor-bearing mice after treatment. Bars represent SEM. **: p<0.01 vs. vehicle control.
20A-20D depict thymus and spleen weights of B16F10 tumor-bearing mice after treatment. Bars represent SEM. *: p<0.05 vs. vehicle control. **: p<0.01 vs. vehicle control.
21 depicts the tumor volume of S180 tumor-bearing mice on treatment. Bars represent SEM. **: p<0.01 vs. vehicle control.
22 depicts tumor weights of S180 tumor-bearing mice after treatment. Bars represent SEM. **: p<0.01 vs. vehicle control.
23 depicts the survival rate of S180 tumor-bearing mice on treatment. ● Saline, ■ CTX, ▲ YS-ON-001.
24A and 24B show chest CT. 24A shows a CT scan prior to administration of YS-ON-001; 24B shows CT scans after two courses of YS-ON-001 treatment.

Example

The invention will now be generally described, which will be more readily recognized by reference to the following examples, which are presented by way of illustration and are not intended to be limiting of the invention.

Unless otherwise stated, animal studies described in this disclosure were performed in accordance with the Law on Laboratory Animals, Guidelines for the Care of Laboratory Animals, and China National Standard GB/14925.

Example 1. Method of making a composition of the present disclosure

1. Virus:

Inactivated rabies virus.

2. A composition of the present disclosure is prepared according to the following ingredients:

The composition comprises inactivated rabies virus, PIC, kanamycin and calcium chloride. The composition further comprises auxiliary substances comprising maltose, dextran and human serum albumin.

Here, inactivated rabies virus: PIC = 2.0 IU: 1000 pg.

Under sterile conditions, the composition is formulated in a physiologically acceptable buffer. The concentration and volume of the composition to be formulated will depend on the subject to be administered (including but not limited to age, sex, weight, health condition), cancer condition (including but not limited to cancer type, severity), route of administration, and administration. It can be adjusted according to factors including frequency. For the same therapeutically effective amount, the dosage volume is small when the concentration of the composition is high; When the concentration of the composition is low, the dosage volume is large.

When the composition is applied to mice, the composition YS-ON-001 is prepared at a concentration of 2.0 IU/mL of inactivated rabies virus, 1000 pg/mL of PIC, 800 IU/mL of kanamycin and 0.16 pmol/mL of calcium ions. .

PIC is unstable in the human body and can be rapidly destroyed by nucleases. Antibiotics (or polyamine compounds) and positive ions can form a three-dimensional structure with PIC, thereby increasing PIC stability. The electron microscopic bran shows the final structure in FIG. 1 .

Example 2. Therapeutic effect of composition (YS-ON-001) in lung cancer animal model

I. Construction of the tumor model

1. Animals

1.1 Female C57BL/6 mice (6 weeks old, body weight 17.5 ± 0.1 g) were obtained from Shanghai SIPPR-BK Laboratory Animal Co., Ltd. Ltd.

1.2 Animal breeding

Animals were fed according to SPF level animal feeding conditions and given a diet (Keaoxieli certified rodent diet). Cages, beds, feed and drinking water were autoclaved. Cages containing 3 to 5 animals per cage were placed in a laminar flow rack with a cleanliness of 100 and the cages were changed twice a week. The temperature of the animal room is 21-25°C, and the relative humidity is 40%-70%.

2. Tumor cell lines

The murine Lewis lung cancer cell line LL/2 (ATCC® CRL-1642 ^™ ) was obtained from the American Type Cultures Collection (ATCC). Cells were maintained in an atmosphere of 5% C0 ₂ at 37° C. in DMEM supplemented with 10% fetal bovine serum. The passage ratio was 1:5 to 1:8 at a frequency of 3 to 4 times per week.

3. Construction of the LL/2 Tumor Graft Model

A LL/2 murine Lewis lung cancer model was established by subcutaneous inoculation with 1× ^{10 6} cells per animal in female C57BL/6 mice. The tumor formation rate was 100%. Three days after cell inoculation, the cell inoculated mice were randomized into treatment groups by body weight. Animals in each group were administered according to Table 1.

II. test method

1. test group

There were four groups: vehicle group, cisplatin group, YS-ON-001 low-dose group, and YS-ON-001 high-dose group.

Experimental design for LL/2 group cure N route of administration Dosage level schedule One vehicle
(negative control) 10 i.m. 0.2 mL/mouse q3d, a total of 6 doses 2 cisplatin
(positive control) 10 i.v. 5 mg/kg QW, a total of 3 doses 3 composition
YS-ON-001 10 i.m. 0.1 mL/mouse q3d, a total of 6 doses 4 composition
YS-ON-001 10 i.m. 0.2 mL/mouse q3d, a total of 6 doses

Group 1: In the vehicle group, DPBS was injected at 0.1 mL/site in both hind limbs.

Group 3: YS-ON-001 low-dose group, 0.1 mL of YS-ON-001 was injected into the right upper leg and 0.1 mL of DPBS was injected into the left hind limb at 0.1 mL/mouse.

Group 4: YS-ON-001 High-dose group, YS-ON-001 was injected into both hind legs at 0.1 mL/site, for a total of 0.2 mL.

2. The study was terminated when the mean tumor volume of the vehicle group reached 2500 mm ³ .

3. At the end of the study, the tumor mass was removed and pictures were taken. Tumor weight was recorded. The thymus and spleen were removed and weighed. Then, the organ index was calculated.

III. Observational indices

1. Tumor Volume

The anti-tumor effect of the test article was observed by measuring the tumor diameter. Tumor volume was measured three times weekly and the relative tumor growth rate T/C (%) was calculated.

1.1 Tumor volume (TV) was calculated as follows:

TV=1/2 xaxb ² ,

In the formula, a and b represent length and width, respectively.

1.2 Relative tumor growth rate T/C (%) = TV _t /TV _c x 100%

where TV _t is the mean tumor volume of the treatment group and TVC is the mean tumor volume of the vehicle control group.

2. Tumor Weight

At the end of the study, the tumor mass was removed and weighed. Tumor weight was recorded and tumor inhibition was calculated according to the following equation:

Tumor inhibition = (TW _C -TW _T )/TW _c x 100%,

where TW _t is the mean tumor weight of the treatment group and TV _C is the mean tumor weight of the control group.

3. Weight

The body weight of each mouse was measured 3 times a week. Change in body weight (BWC) was calculated as follows:

BWC = (BW _n - BW ₀ )/BW ₀ x 100%

where BW _n is the BW on day n and BW ₀ is the BW on the day the treatment started.

4. Thymus and Spleen Weights

At the end of the study, the thymus and spleen were removed and weighed. The bronchial index was calculated as follows: tracheal index = tracheal weight/body weight x 100%.

IV. Data provision and analysis

Data were reported as mean±SEM. Comparisons between groups were analyzed using ANOVA.

V. Results

1. Tumor Volume

The effect of YS-ON-001 on LL/2 murine Lewis lung tumor volume is graphically shown in FIG. 2A .

The effect of YS-ON-001 on the proliferation of LL/2 murine Lewis lung tumors is graphically shown in FIG. 2B .

The day of tumor is set as day 0. On day 3, animals were randomized by body weight and dosed according to group assignment.

Tumors in the vehicle control group grew steadily in all 10 mice. At the end of the study, the mean tumor volume reached 2707±257 mm ³ .

Treatment with cisplatin significantly inhibited tumor growth starting from day 10 until the end of the study, resulting in a relative tumor proliferation rate of 53.04%, which was significantly different compared to the vehicle control group ( p < 0.01). .

YS-ON-001 treatment significantly inhibited tumor growth starting from day 10 until the end of the study compared to the vehicle control group (p < 0.01). Although the tumor volume in the high-dose group was lower than that in the low-dose group, the difference was not significant (p > 0.05). At the end of the study, the relative tumor growth rates were 43.36% and 34.33%, respectively.

2. Tumor Weight

The effect of YS-ON-001 on the weight of Lewis lung tumors in LL/2 murine is shown graphically in FIG. 3 .

At the end of the study, the tumor mass was removed and weighed. Similar to tumor volume, treatment with cisplatin or YS-ON-001 significantly lowered tumor weight compared to vehicle control group ( p < 0.01). Tumor weights in the YS-ON-001 high-dose group were significantly lower than those in the cisplatin group and in the YS-ON-001 low-dose group ( p <0.05). The inhibition rates were 29.46%, 29.49% and 53.87% for the cisplatin group, YS-ON-001 low-dose and high-dose groups, respectively.

3. Weight

The effect of YS-ON-001 on body weight and body weight change of LL/2 murine Lewis lung tumor is shown graphically in FIG. 3 .

Mean body weight (BW) was significantly affected by treatment with cisplatin compared to vehicle group on day 5 and from day 10 to the end of the study ( p <0.01). The same results were observed for changes in body weight.

Mouse body weight was clearly unaffected by treatment in both YS-ON-001 groups compared to vehicle group ( p >0.05). Body weight change in the YS-ON-001 high-dose group was significantly lower on day 7 compared to the vehicle control group ( p <0.05), while no significant change was observed at other time points.

4. Thymus and Spleen Weights

Thymic and spleen weights as well as bronchial indices of LL/2 murine Lewis lung tumors are graphically shown in FIGS. 6 and 7 .

At the end of the study, tumor masses were excluded, and the thymus and spleen were removed and weighed. Compared with the vehicle group, cisplatin treatment significantly reduced the weight of the thymus and the weight of the spleen. Thymus weight and thymus index were significantly decreased compared to that in the vehicle control group ( p <0.01). The spleen weight was significantly reduced compared to that in the vehicle control group ( p <0.05), but the spleen index showed a trend to decrease, which was not significant (p>0.05). Thymus weight, spleen weight and their bronchial index were not affected by treatment with YS-ON-001 compared to vehicle group.

VI. Discussion and Conclusion

In this example, the vehicle control group showed strong tumor growth and the cisplatin group showed a significant inhibitory effect on tumor growth as a positive control.

YS-ON-001 showed anti-tumor activity in a dose-dependent manner when used as a single agent at 0.1 mL/mouse and 0.2 mL/mouse.

Moreover, the animals were well tolerated and showed no significant effect on body weight, spleen weight and thymus weight. Adverse events were significantly lower than those in the cisplatin group. The antitumor effect in the YS-ON-001 high-dose group was better than that in the cisplatin group.

The results showed that YS-ON-001 exerted an inhibitory effect on tumor growth in a dose-dependent manner at the dose levels tested in the LL/2 Lewis lung cancer animal model. On the other hand, tumor-bearing mice did not show any obvious side effects.

Example 3. Therapeutic effect of the composition (YS-ON-001) in an animal model of breast cancer

I. Construction of the tumor model

1. Animals

1.1 Female Balb/c mice (6 weeks old, body weight 17.2 ± 0.1 g) were obtained from Shanghai SIPPR-BK Laboratory Animal Co. Ltd.

2.2. animal breeding

Animals were fed according to SPF level animal feeding conditions and given a diet (Keaoxieli certified rodent diet). Cages, dragonflies, feed and drinking water were autoclaved. Cages containing 3 to 5 animals per cage were placed in a laminar flow rack with a cleanliness of 100 and the cages were changed twice a week. The temperature of the animal room is 21-25°C, and the relative humidity is 40%-70%.

2. Tumor cell lines

The murine breast cancer cell line 4T1 was obtained from the American Type Cultures Collection (ATCC) and maintained by HD Biosciences. Cells were maintained in an atmosphere of 5% C0 ₂ at 37° C. in DMEM supplemented with 10% fetal bovine serum. The passage ratio was 1:5 to 1:8 at a frequency of 3 to 4 times per week.

3. Construction of the 4T1 Tumor Transplantation Model

A 4T1 murine mammary tumor model was established by subcutaneous inoculation in female Balb/c mice at 1× ^{10 6} cells per animal. The tumor formation rate was 100%. Three days after cell inoculation, the cell inoculated mice were randomized into treatment groups by body weight. Animals in each group were administered according to Table 2.

Experimental design for 4T1 group cure N route of administration Dosage level schedule One vehicle
(negative control) 10 i.m. 0.2 mL/mouse q3d, a total of 6 doses 2 docetaxel
(positive control) 10 i.v. 10 mg/kg QW, a total of 3 doses 3 composition
YS-ON-001 10 i.m. 0.1 mL/mouse q3d, a total of 6 doses 4 composition
YS-ON-001 10 i.m. 0.2 mL/mouse q3d, a total of 6 doses

Group 1: In the vehicle group, DPBS was injected at 0.1 mL/site in both hind limbs.

Group 3: YS-ON-001 low-dose group, 0.1 mL of YS-ON-001 was injected into the right upper leg and 0.1 mL of DPBS was injected into the left hind limb at 0.1 mL/mouse.

Group 4: YS-ON-001 High-dose group, YS-ON-001 was injected into both hind legs at 0.1 mL/site, for a total of 0.2 mL.

II. test method

1. test group

There were four groups: vehicle group, docetaxel group, YS-ON-001 low-dose group, and YS-ON-001 high-dose group.

2. The study was terminated when the mean tumor volume of the vehicle group reached 2500 mm ³ .

3. At the end of the study, the tumor mass was removed and pictures were taken. Tumor weight was recorded. The thymus and spleen were removed and weighed. Then, the organ index was calculated.

III. Observational indices: same as in section III of Example 2.

IV. Data provision and analysis

Data were reported as mean±SEM. Comparisons between groups were analyzed using ANOVA.

V. Results

1. Tumor Volume

The effect of YS-ON-001 on 4T1 murine breast tumor volume is graphically shown in FIG. 8 .

The effect of YS-ON-001 on the proliferation of 4T1 murine mammary tumors is shown graphically in FIG. 9 .

The day of tumor is set as day 0. On day 3, animals were randomized by body weight and dosed according to group assignment.

Tumors in the vehicle control group grew steadily in all 10 mice. At the end of the study, the mean tumor volume reached 2576±108 mm ³ .

Treatment with docetaxel significantly inhibited tumor growth from day 8 until the end of the study, resulting in a relative tumor growth rate of 50.12%, which was significantly different compared to the vehicle control group ( p < 0.01). .

YS-ON-001 treatment significantly inhibited tumor growth starting from day 8 until the end of the study compared to the vehicle control group (p < 0.01). There was a significant difference between the YS-ON-001 low-dose group and the high-dose group from day 11 to the end of the study (p<0.05). At the end of the study, the relative tumor growth rates were 63.43% and 45.87%, respectively.

2. Tumor Weight

The effect of YS-ON-001 on the weight of 4T1 murine mammary tumors is shown graphically in FIG. 10 .

At the end of the study, the tumor mass was removed and weighed. Similar to tumor volume, treatment with docetaxel significantly lowered tumor weight compared to the vehicle control group ( p < 0.01). The YS-ON-001 high-dose group significantly reduced tumor weight when compared to the vehicle control group ( p < 0.01). The YS-ON-001 low-dose group slightly lowered the tumor weight when compared to the vehicle control group. The tumor weights in the YS-ON-001 high-dose group were significantly lower than those in the YS-ON-001 low-dose group ( p < 0.05). The inhibition rates were 35.55%, 18.21%, and 42.26% for the docetaxel group, YS-ON-001 low-dose and high-dose groups, respectively.

3. Weight

The effect of YS-ON-001 on body weight and body weight change of 4T1 murine breast tumors is shown graphically in FIGS. 11 and 12 .

Mean body weight (BW) was significantly affected by treatment with docetaxel compared to vehicle group on day 8 and from day 13 to the end of the study ( p <0.01). Mean body weight change was significantly reduced by treatment with docetaxel compared to the vehicle group from day 4 to the end of the study ( p <0.05). Body weight at the end of the study also showed a downward trend when divided into groups compared to that of the same group.

Mouse body weight was clearly not affected in the YS-ON-001 low-dose group compared to the vehicle group. The body weight of the YS-ON-001 high dose group was significantly lower from day 15 than the body weight in the vehicle group ( p >0.05). Body weight change in both YS-ON-001 groups was significantly lower on day 4 and from day 15 to the end of the study compared to the vehicle control group (p<0.05 for the low-dose group and p<0.05 for the high-dose group) p<0.01). However, the body weight was still increased compared to the body weight in the same group at the time of dividing the groups.

4. Thymus and Spleen Weights

Thymic and spleen weights as well as bronchial indices of 4T1 murine breast tumors are graphically shown in FIGS. 13 and 14 .

At the end of the study, tumor masses were excluded, and the thymus and spleen were removed and weighed.

Compared with the vehicle group, docetaxel treatment significantly reduced thymus and spleen weights and thymus and spleen indices ( p <0.01). Thymus weight, spleen weight and thymic bronchial index were not affected by treatment with YS-ON-001 compared to vehicle group. At both doses of the YS-ON-001 group, the spleen trachea index significantly increased compared to the vehicle group ( p <0.01 for the low-dose group, p <0.05 for the high-dose group).

VI. Discussion and Conclusion

In this example, the vehicle control group showed strong tumor growth and the docetaxel group showed a significant inhibitory effect on tumor growth as a positive control.

YS-ON-001 showed anti-tumor activity in a dose-dependent manner when used as a single agent at 0.1 mL/mouse and 0.2 mL/mouse. Moreover, the animals were well tolerated and showed no significant effect on thymus weight. The antitumor effect of the high-dose group was similar to that of the docetaxel group, and the side effects were significantly reduced.

In this example, the spleen index in both YS-ON-001 groups increased compared to that in the vehicle control group. Further analysis is needed to understand why. Preliminary assessment may improve immune function resulting in weight gain in immune organs.

The results showed that YS-ON-001 exerted an inhibitory effect on tumor growth in a dose-dependent manner at the dose levels tested in the 4T1 breast cancer animal model. On the other hand, tumor-bearing mice did not show any obvious side effects.

Example 4. Therapeutic effect of composition (YS-ON-001) alone or in combination with sorafenib in subcutaneous H22 murine hepatocellular carcinoma model

I. Construction of the tumor model

1. Animals and animal breeding were the same as in Example 2.

2. Tumor cell lines

The murine hepatocellular carcinoma cell line H22 was obtained from the China Center for Type Culture Collection (CCTCC) and maintained by HD Biosciences. Cells were maintained in Balb/c mice as ascites. The passage interval is 7-8 days.

3. Construction of the H22 Tumor Graft Model

Donor mice with H22 ascites were sacrificed and sterilized by immersion in 75% alcohol. Ascites was collected and resuspended to the appropriate density in pre-chilled DPBS. A H22 murine hepatocellular carcinoma tumor model was established by subcutaneous inoculation in female Balb/c mice at 5×10 ⁶ cells per animal. The tumor formation rate was 100%. Three days after cell inoculation, the cell inoculated mice were randomized into treatment groups by body weight. Animals in each group were administered according to Table 3.

II. test method

1. test group

There were four groups: vehicle group, sorafenib group, YS-ON-001 group, and sorafenib+YS-ON-001 group.

Experimental group and dose group cure# N Route of Administration# Dosage level schedule One vehicle* 10 i.m./s.c 0.2 mL/mouse BID 2 sorafenib 10 p.o. 60 mg/kg QD 3 composition
YS-ON-001 10 i.m./s.c 0.2 mL/mouse BID 4 Sorafenib + composition
YS-ON-001 10 po + imls.c 60 mg/kg +
0.2 mL/mouse BID + QD

*: Vehicle is DPBS.

#: Sorafenib was administered via oral feeding, vehicle and YS-ON-001 were injected i.m. 0.1 mL and s.c. 0.1 mL.

2. The study was terminated when the mean tumor volume of the vehicle group reached 2500 mm ³ .

3. At the end of the study, the tumor mass was removed and pictures were taken. Tumor weight was recorded.

III. Observational index: same as in Example 2.

IV. result

1. Tumor Volume

The effect of YS-ON-001 on H22 murine hepatocellular carcinoma tumor volume is graphically shown in FIG. 15A .

The effect of YS-ON-001 on H22 murine hepatocellular carcinoma tumor proliferation is graphically shown in FIG. 15B .

The day of tumor is set as day 0. On day 3, animals were randomized by body weight and dosed according to group assignment.

Tumors in the vehicle-treated group grew steadily in all 10 mice. When the vehicle group was terminated on day 12, the mean tumor volume reached 2120±182 mm ³ .

One mouse in the vehicle group and two mice in the sorafenib group were sacrificed on days 10 and 19 when their tumor volume reached 2500 mm ³ . The entire study was terminated on day 21.

Treatment with sorafenib significantly inhibited tumor growth (p<0.01) compared to that in the vehicle control group from day 3 to the end of the study. The relative tumor growth rate at day 12 is 29.90%.

Treatment with sorafenib+YS-ON-001 significantly inhibited tumor growth from day 1 to the end of the study compared to that in the vehicle control group (p<0.01). The relative tumor growth rate at day 12 is 10.61%.

There was a significant difference (p<0.05 or p<0.01) in tumor volume between the sorafenib group and the sorafenib+YS-ON-001 group from days 1, 5, and 10 to the end of the study.

There was a significant difference (p<0.05 or p<0.01) in tumor volume between the YS-ON-001 and sorafenib+YS-ON-001 groups from days 5 and 10 to the end of the study.

V. DISCUSSION AND CONCLUSION

In this example, the vehicle control group showed strong tumor growth and the sorafenib group showed a significant inhibitory effect on tumor growth as a positive control. These indicate that the results are reliable.

YS-ON-001 showed potent anti-tumor activity when 0.2 mL/mouse of BID was used. Sorafenib+YS-ON-001 showed a very significant inhibition of tumor growth and a significant synergistic effect. There were no animal deaths due to drug toxicity in either the single drug group or the combination drug group.

The results showed that YS-ON-001 and the combination group showed strong anti-tumor activity in the H22 murine hepatocellular carcinoma tumor mouse model at the dose levels tested. Combination of YS-ON-001 with sorafenib induced significantly improved antitumor activity compared to using either agent alone.

Example 5. Therapeutic effect of composition (YS-ON-001) and PIKA alone in subcutaneous LL/2 murine Lewis lung cancer model in female C57BL/6 mice

I. Construction of the tumor model

1. Animals and animal breeding were the same as in Example 2.

2. The murine Lewis lung cancer cell line LL/2 was obtained and cultured, and the LL/2 murine Lewis lung tumor model was established in the same manner as in Example 2. Animals in each group were administered according to Table 4.

II. test method

1. test group

There were 6 groups: vehicle group, cisplatin group, YS-ON-001 group, YS-ON-002 (no virus) group, rabies vaccine group and cisplatin+YS-ON-001 group. The difference between YS-ON-001 and YS-ON-002 is that YS-ON-002 has no inactivated rabies virus.

Experimental group and dose group cure N route of administration Dosage level* schedule One vehicle 10 i.m. 0.2 mL/mouse Q2D, total 7 times 2 cisplatin 10 i.v. 5 mg/kg QW, total of 2 times 3 composition
YS-ON-001 10 i.m. 0.2 mL/mouse Q2D, total 7 times 4 YS-ON-002
(PIKA only) 10 i.m. 0.2 mL/mouse Q2D, total 7 times 5 Rabies Vaccine (Vero Cells) 10 i.m. 0.2 mL/mouse Q2D, total 7 times 6 cisplatin + composition
YS-ON-001 10 iv + im 0.2 mL/mouse QW (2 times in total) + Q2D (7 times in total)

*: In the vehicle group, DPBS was injected at 0.1 mL/site in both hind limbs. YS-ON-001 and YS-ON-002 were injected into both hind legs at 0.1 mL/site.

2. The study was terminated when the mean tumor volume of the vehicle group reached 2500 mm ³ .

3. At the end of the study, the tumor mass was removed and pictures were taken. Tumor weight was recorded.

III. Observational indices

1. Tumor Volume

Measurements and calculations of tumor volume and tumor weight were the same as in Example 2.

2. Combination drug index Q values were calculated according to Jin's formula, Q = 0.85-1.15 for additive effects and Q > 1.15 for synergistic effects.

Q=Ea+b/(Ea+Eb-EaxEb)

where Ea+b is the rate of tumor suppression in the drug combination group, and Ea and Eb are the rates of tumor suppression in the single drug group.

IV. result

1. Tumor Volume

The effects of YS-ON-001, YS-ON-002 and combination groups on tumor volume in mice bearing LL/2 murine Lewis lung carcinoma are graphically presented in FIG. 16A .

The effects of YS-ON-001, YS-ON-002 and combination groups on tumor proliferation in mice bearing LL/2 murine Lewis lung carcinoma are graphically shown in FIG. 16B .

The day of tumor is set as day 0. On day 3, animals were randomized by body weight and dosed according to group assessment.

Tumors in the vehicle control group grew steadily in all 10 mice. When the vehicle group was terminated, the mean tumor volume reached 2535 ± 148 mm ³ .

Treatment with cisplatin significantly inhibited tumor growth compared to the vehicle control group from day 5 to the end of the study (p<0.01), resulting in a relative tumor proliferation rate of 47.46%.

Treatment with YS-ON-001 and YS-ON-002 significantly inhibited tumor growth (p<0.01) when compared to the vehicle control group from day 5 to the end of the study. The relative tumor growth rates were 37.02% and 40.56%, respectively.

Treatment with the rabies vaccine showed no apparent effect compared to the vehicle control group (p>0.05). The relative tumor growth rate was 97.87% at the end of the study.

The combination group significantly inhibited tumor growth (p<0.01) when compared to the vehicle control group from day 5 to the end of the study. The relative tumor growth rate was 28.38% at the end of the study. The relative tumor growth rate was lower than that in the cisplatin and YS-ON-001 groups alone. According to Jin's formula, YS-ON-001 and cisplatin showed an additional effect, Q=0.87.

The tumor volume of the YS-ON-001 group was lower than that of the YS-ON-002 group, but the difference was statistically insignificant. The tumor volume in the YS-ON-001 group was lower than in the YS-ON-002 group from day 5 to the end of the study.

According to Jin's formula, the rabies virus and other components in the composition show an additional effect, Q=0.93.

2. Tumor Weight

The effects of YS-ON-001, YS-ON-002 and combination groups on tumor weight in mice bearing LL/2 murine Lewis lung carcinoma are graphically shown in FIG. 17 .

At the end of the study, the tumor mass was removed and weighed. Similar to the effect on tumor volume, the cisplatin, YS-ON-001, YS-ON-002 and combination groups had significantly reduced tumor weight compared to the vehicle control group ( p <0.01), each 42.38% , showed tumor suppression rates of 60.88%, 56.04% and 75.44%.

According to Jin's formula, the rabies virus and other components in the composition show an additional effect, Q=0.97.

V. Discussion and Conclusion

In this example, the vehicle control group showed strong tumor growth and the cisplatin group showed a significant inhibitory effect on tumor growth as a positive control. These indicate that the results are reliable.

YS-ON-001 and YS-ON-002 exhibited strong anti-tumor activity when used as a single agent at 0.2 mL/mouse administered i.m. in the LL/2 murine Lewis lung tumor model. The animals were well tolerated and showed some effect on body weight, but the toxic side effects were significantly lower than that of the control drug cisplatin. The anti-tumor effect was comparable to that of the control drug cisplatin.

The relative tumor growth rate of the combination group was 28.38%, which was lower than that of the groups treated with cisplatin and YS-ON-001 alone. According to Jin's formula, YS-ON-001 and cisplatin showed additional effects.

Although there is no statistically significant change in the tumor suppression effect between YS-ON-001 and YS-ON-002, the rabies virus and other components in the composition show additional effects according to Jin's formula.

The results showed that YS-ON-001 and YS-ON-002 had inhibitory effects at the dose levels tested in the LL/2 Lewis lung cancer animal model. Composition YS-ON-001 and cisplatin showed additional effects.

Example 6. Therapeutic effect of composition (YS-ON-001) in B16F10 tumor metastasis model in female C57BL/6 mice

I. Construction of the tumor model

1. Animals and animal breeding were the same as in Example 2.

2. Tumor cell lines

Cell line B16F10 was obtained from the American Type Cultures Collection (ATCC) and maintained by HD Biosciences. Cells were maintained in an atmosphere of 5% C0 ₂ at 37° C. in DMEM supplemented with 10% fetal bovine serum. The passage ratio was 1:5 to 1:8 at a frequency of 3 to 4 times per week.

3. Construction of the LL/2 Tumor Transplantation Model

A B16F10 metastatic tumor model was established by intravenous inoculation in female C57BL/6 mice at 5×10 ⁴ /0.2 mL per animal. One day after cell inoculation, mice were randomized into treatment groups by body weight. Animals in each group were administered according to Table 5.

II. test method

1. test group

There were three groups: the vehicle group, the cisplatin group and the YS-ON-001 group.

Experimental group and dose group cure N Route of Administration# Dosage level schedule One vehicle* 10 i.m./s.c 0.2 mL/mouse/dose BID 2 cisplatin 10 i.v. 5 mg/kg Q4D 3 composition
YS-ON-001 10 i.m./s.c 200 mL/mouse/dose BID

*: The vehicle is DPBS.

#: i.m. to the animals in the vehicle group and the YS-ON-001 group, respectively. (Hind legs) and s.c. 0.1 mL was administered via injection.

2. At the end of the study, the study was terminated on day 15. The lungs were removed and pictures were taken. The number of metastatic spots was counted. The thymus and spleen were removed, weighed and calculated for the duration index.

III. Observational indices

1. weight

The body weight of each mouse was measured 3 times a week. Change in body weight (BWC) was calculated as follows:

BWC = (BW _n - BW _o )/BW ₀ x 100%

where BW _n is the BW on day n and BW ₀ is the BW on the day the treatment started.

2. Number of lung metastases

At the end of the study, the animals were sacrificed and the lungs removed. Then, the number of transitions was calculated.

3. Thymus and Spleen Weights

At the end of the study, the thymus and viscera were removed and weighed.

IV. Data provision and analysis

Data were reported as mean±SEM. Comparisons between groups were analyzed using ANOVA.

V. Results

1. weight

The effect of YS-ON-001 on the body weight and body weight change of mice is shown graphically in FIGS. 18A and 18B .

Mean body weight (BW) was significantly lowered by treatment with cisplatin compared to the vehicle group (p<0.01) from day 5 to the end of the study (excluding day 7). Similar results were also observed for changes in body weight.

In the YS-ON-001 group, body weight temporarily decreased slightly compared to the vehicle control group. However, on days 5 and 15, the body weight in the YS-ON-001 group was significantly lowered compared to the vehicle control group.

2. Number of lung metastases

The effect of YS-ON-001 on the number of lung metastases in mice is graphically shown in FIG. 19 .

At the end of the study, the animals were sacrificed and the lungs removed. The number of transitions was then counted.

Compared with the vehicle group, the number of lung metastases was significantly decreased by treatment with cisplatin ALC YS-ON-001 ( p <0.01).

3. Thymus and Spleen Weights

The thymus and spleen weights as well as the respective bronchial indices of the mice are graphically displayed and shown in FIG. 20 .

At the end of the study, in addition to the lungs, the thymus and spleen were removed and weighed. Institutional indices were calculated for each institution.

Compared with the vehicle group, treatment with cisplatin significantly reduced thymus weight ( p <0.01), thymus index ( p <0.01), while spleen weight and spleen index were unaffected.

Compared with the vehicle group, thymus weight and thymus index were significantly decreased ( p <0.01) by treatment with YS-ON-001, while spleen weight and spleen index were significantly increased ( p < 0.01).

VI. Discussion and Conclusion

In this example, lung metastases were observed in all animals in the vehicle control group, so the mean number of lung metastases was 93.43±1.76. The mean number of lung metastases was significantly decreased in the cisplatin group. These indicate that the results are reliable.

YS-ON-001 exhibited strong anti-metastatic activity, with the mean number of lung metastases being significantly reduced in the B16F10 tumor metastasis model at the dose levels tested.

Example 7. Therapeutic Effect of Composition (YS-ON-001) in S180 Tumor Model in Female Balb/C Mice

I. Construction of the tumor model

1. Animals and animal breeding were the same as in Example 2.

2. Tumor cell lines

Cell line S180 was obtained from the Cell Bank of Chinese Academy of Medical Sciences and maintained in the IPE-CAS laboratory. Cells were maintained as ascites in Balb/c mice. The passage interval is 5-6 days.

3. Construction of the S180 Tumor Graft Model

Balb/c animals were fed for 5-7 days, and S180 tumors were inoculated intraperitoneally into F0 generation mice. After the ascites was taken when the abdominal cavity reached a certain size, it was inoculated into F1 generation mice. For the test, mice of the F2-F3 generation were used. The S180 tumor model was established by subcutaneous inoculation with a cell suspension (2x10 ⁶ /0.2 mL/animal) in female Balb/c mice. Two days after cell inoculation, the cell inoculated mice were randomized into treatment groups. Animals in each group were administered according to Table 6.

II. test method

1. test group

There were three groups: the vehicle group, the cyclophosphamide (CTX) group, and the YS-ON-001 group.

Experimental group and dose group cure N route of administration Dosage level schedule One plain saline 10 i.m. 0.2 mL/mouse/dose Q2D, total of 10 episodes 2 Cyclophosphamide (CTX) 10 i.p. 20 mg/kg Q2D, total of 10 episodes 3 composition
YS-ON-001 10 i.m. 0.2 mL/mouse/dose Q2D, total of 10 episodes

2. At the end of the study, the study was terminated on day 21. The tumor was removed and pictures were taken. The weight of the tumor was recorded.

III. Observational indices

1. Tumor Volume

The anti-tumor effect of the test article was observed by measuring the tumor diameter. Tumor volume was measured three times a week and the relative tumor growth rate T/C (%) was calculated.

1.1 Tumor volume (TV) was calculated as follows:

TV=1/2 xaxb ² ,

In the formula, a and b represent length and width, respectively.

2. At the end of the study, the tumor mass was removed and weighed.

IV. Data provision and analysis

Data were reported as mean±SEM. Comparisons between groups were analyzed using ANOVA.

V. Results

1. Tumor Volume

The effect of the YS-ON-001 group on the tumor volume of the S180 tumor model in female Balb/C mice is graphically shown in FIG. 21 .

In the saline-treated control group, tumors grew steadily in all 10 mice. On day 21, the mean tumor volume reached 2000 mm ³ .

Treatment with CTX significantly inhibited tumor growth compared to the saline control group from day 4 to the end of the study ( p <0.01).

Treatment with YS-ON-001 significantly inhibited tumor growth when compared to the saline control group from day 4 to the end of the study ( p <0.01). The tumor volume in the YS-ON-001 group was similar to that in the CTX group.

2. Tumor Weight

The effect of the YS-ON-001 group on the tumor weight of the S180 tumor model in female Balb/C mice is graphically shown in FIG. 22 .

At the end of the study, the tumor mass was removed and weighed. Similar to the effect on tumor volume, CTX and YS-ON-001 significantly reduced tumor weight when compared to the saline control group (p<0.01).

VI. Discussion and Conclusion

In this example, the vehicle control group showed strong tumor growth and the CTX group showed a significant inhibitory effect on tumor growth as a positive control.

Treatment with YS-ON-001 significantly inhibited tumor growth while significantly reducing tumor volume and tumor weight.

The results showed that YS-ON-001 exhibited strong anti-tumor activity in the S180 tumor model at the dose levels tested.

Example 8. Therapeutic effect of composition (YS-ON-001) in Ehrlich ascites cancer (EAC) tumor model in female Balb/C mice

I. Construction of the tumor model

1. Animals

1.1 Female Kunming mice (5-6 weeks old) were obtained and tested after adaptive feeding for 5-6 days.

1.2 Animal breeding

Animals were fed according to SPF level animal feeding conditions and given a diet (Keaoxieli certified rodent diet). Cages, dragonflies, feed and drinking water were autoclaved. Cages containing 3 to 5 animals per cage were placed in a laminar flow rack with a cleanliness of 100 and the cages were changed twice a week. The temperature of the animal room is 21-25°C, and the relative humidity is 40%-70%.

2. Tumor cell lines

The Ehrlich Ascites Cancer (EAC) tumor cell line EAC was obtained from the Cell Bank of Chinese Academy of Medical Sciences and maintained in the IPE-CAS laboratory. Cells were maintained as ascites in Kunming mice. The passage interval is 7-8 days.

3. Construction of the tumor transplantation model

Animals were fed for 5-7 days and EAC tumors were inoculated intraperitoneally into F0 generation mice. After the ascites was taken when the abdominal cavity reached a certain size, it was inoculated into F1 generation mice. For the test, mice of the F2-F3 generation were used. EAC tumor models were established by intraperitoneal inoculation with cell suspension (1x10 ⁶ /0.2 mL/animal) in female Kunming mice. Two days after cell inoculation, the cell inoculated mice were randomized into treatment groups. Animals in each group were administered according to Table 7.

Experimental design for EAC group cure N route of administration Dosage level schedule One vehicle 10 i.m. 0.2 mL/mouse/dose Q2d, total of 10 2 CTX 10 i.m. 20 mg/kg Q2d, total of 10 3 composition
YS-ON-001 10 i.m. 0.2 mL/mouse/dose Q2d, total of 10

Group 1: In the vehicle group, PBS was injected into both hind legs at 0.1 mL/site.

Group 3: YS-ON-001 group, YS-ON-001 was injected into both hind limbs at 0.1 mL/site, a total of 0.2 mL.

II. test method

1. test group

There were three groups: the vehicle group, the CTX group, and the YS-ON-001 group.

2. At the end of the study, the study was terminated on day 21. The tumor was removed and pictures were taken. The weight of the tumor was recorded.

III. Observational indices: duration, tumor growth retardation

Tumor growth delay (TGD) was calculated as (TGD) = (T-C)/C x 100%, where T and C are the median duration of individual mice to death in days for treatment and control groups, respectively, in days. indicates.

IV. Data provision and analysis

Comparisons between groups were analyzed using ANOVA.

V. Results

The survival rate of the EAC tumor model in female Kunming mice is graphically shown in FIG. 23 .

The day of tumor inoculation is set as day 0. On day 2, animals were randomized and dosed according to group assessment. Tumors in the vehicle control group grew steadily in all 10 mice. Mice were observed to die on day 23, and all mice died on day 27. The median duration was 25.5 days.

Compared to the vehicle group, mice in the CTX group were observed to die on day 27, and all mice died on day 30. Tumor growth retardation (TGD) when treated with CTX was 14.74%, with a median duration of 29 days.

Compared to the vehicle group, mice in the YS-ON-001 group were observed to die on day 33, and all mice died on day 39. Tumor growth retardation (TGD) when treated with CTX was 38.65%, with a median duration of 34.5 days.

VI. Discussion and Conclusion

In this example, the vehicle control group showed strong tumor growth and the CTX group showed significant tumor growth retardation as a positive control.

Treatment with the composition YS-ON-001 at 0.2 mL/mouse had a significant tumor growth retardation effect, while the animals were well tolerated. The anti-tumor effect of YS-ON-001 is significantly better than that of the CTX control drug.

The results showed that YS-ON-001 had anti-tumor activity and inhibitory effects on tumor growth in the EAC tumor model at the dose levels tested, while tumor-bearing mice were well tolerated.

Example 9. Therapeutic effect of the composition on thyroid cancer including lung and lymph node metastasis

1. Subject:

Mao, a 53-year-old woman, was diagnosed with thyroid cancer that had metastasized to the lungs in August 2013 and underwent radical thyroidectomy. Postoperative pathology revealed papillary carcinoma on the left lobe of the thyroid gland, lymph node metastasis, and nodular goiter on the right lobe of the thyroid gland. Then, on October 28, 2013 and February 6, 2014, she received two doses of iodine-131. In 2015, she received 4 cycles of etoposide + carboplatin radiotherapy (September 28, 2015, October 20, 2015, 11.07. 2015, 12.04 2015). After treatment, 1-2 lesions in the lungs contracted, but other 5-6 lesions did not change, side effects of chemotherapy were evident, and health was poor.

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

With the consent of the patient, YS-ON-001 (1 ml) was administered by intramuscular injection in the upper arm once every 3 days, for a total of 12 doses.

4. Results:

In this example, the subject administered the composition of the present disclosure did not show any obvious side effects, all lesions were stable, and the state of health was also improved.

Example 10. Therapeutic effect of the composition on renal cancer

1. Subject:

Ma, a 70-year-old male, started experiencing intermittent hematuria and occasional pain in her left lower back in November 2015. A comprehensive examination was performed and he was diagnosed with left kidney cancer measuring approximately 11 x 12 x 6 cm. Chest CT showed right lung cancer and chronic inflammation of both lungs, which could be related to liver cyst. In consideration of the patient's age and family opinion, it was decided not to perform surgery or radiotherapy and chemotherapy. He was discharged after brief anti-inflammatory treatment.

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

With the consent of the patient and his family, YS-ON-001 (2 ml) was administered via intramuscular injection in the upper arm, once every 3 days from December 25, for a total of 12 doses.

4. Results:

In this example, the subject administered the composition of the present disclosure did not show any obvious side effects, and the symptoms of hematuria and back pain disappeared.

Example 11. Therapeutic effect of the composition on gastric adenocarcinoma including liver cancer metastasis

1. Subject:

Nan, a 55-year-old woman, was diagnosed with stage IV gastric adenocarcinoma that had metastasized to liver cancer in October 2015, and was treated with oxaliplatin + lapatinib chemotherapy. However, chemotherapy was discontinued due to significant liver damage. After receiving transaminase reduction treatment and symptomatic treatment, liver function returned to basically normal.

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

3.1 First course of treatment:

With the patient's consent, YS-ON-001 (1 ml) was administered once every 3 days from December 25, 2015 via intramuscular injection in the upper arm. The process was stopped on December 29 due to abnormal transaminase elevation.

3.2 Second course of treatment:

On January 2, 2016, liver function was restored to pre-injection levels. The second course of YS-ON-001 treatment was resumed on January 11, and the dose remained the same. Reviewed on January 27th.

4. Results:

After the first course of treatment, transaminase was elevated abnormally, suspected of liver tissue necrosis induced by administration of the composition, and was considered to be a non-specific anti-tumor response.

After the second course of treatment, the patient showed no obvious side effects. Hepatic CT lesions showed reduced nodular shading, proving conjecture of the first course of treatment.

Example 12. Therapeutic effect of composition on breast cancer including lymph, lung and bone metastases

1. Subject:

A 34-year-old woman was diagnosed with invasive ductal carcinoma of the left breast in May 2008 and underwent modified mastectomy. In this case, lymph node metastasis was suspected, and the immunohistochemical results showed ER-, PR++, and HER2++. In June 2015, it was confirmed that it had metastasized to the lungs and bones. Cranial CT showed multiple metastases in bilateral cerebellar hemispheres and cerebellar bursae. The condition was judged to be disease-progressive disease (PD).

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

3.1 First course of treatment:

With the consent of the patient, from September 29, 2015, YS-ON-001 continued through intramuscular injection, once a week (2 ml for the 1st, 3rd, 5th dose, 2nd, 4th, and 6th doses) The dose was 1 ml) and chemotherapy was performed according to the doctor's recommendation.

3.2 Second course of treatment:

From December 14th, YS-ON-001 will be administered by intramuscular injection once every 3 days (1 ml).

4. Results:

After the first course of treatment, as reviewed by chest CT, the right axillary lymph nodes were swollen, the pleural effusion was significantly reduced, the right lung tissue recovered, necrosis occurred in both lung tissues, and brain metastasis was observed. was found to have disappeared. After one week, disease was determined to be stable disease (SD).

After the second course of treatment, nodular shading in both lungs was clearly reduced as reviewed by chest CT. Fig. 24A shows the CT before using YS-ON-001, and Fig. 24B shows the CT after treatment with YS-ON-001 twice. Lung nodule shading was significantly reduced as a result of comparing the two. The compositions of the present disclosure exhibit significant anti-tumor effects.

Example 13. Therapeutic effect of the composition on tongue cancer

1. Subject:

Zhou, a 78-year-old woman, was diagnosed with tongue cancer in April 2015 and underwent an enlargement of the left tongue cancer + partial mandibular resection + extractions. The postoperative pathology showed well-differentiated squamous cell carcinoma. In May 2016, the left tongue cancer recurred, and an enlarged resection was performed for the left recurrent tongue carcinoma.

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

With the patient's consent, from July 26, 2016, YS-ON-001 was administered in 2 doses per day through intramuscular injection of the hip. After dosing for 2 days, the dose was increased to 4 times daily.

4. Results:

In this example, subjects administered the compositions of the present disclosure did not show any apparent side effects. The patient reported a shallow upper jaw ulcer, improved health, better sleep, increased appetite, and improved physical fitness.

Example 14. Therapeutic effect of the composition on prostate cancer including multiple bone metastases

1. Subject:

Xu, an 81-year-old male, was diagnosed with prostate cancer with multiple bone metastases in January 2013.

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

With the consent of the patient, starting from July 3, 2016, YS-ON-001 was administered by intramuscular injection every other day, in 2 doses each time. From August 3, 2016, the capacity was increased to 3. From August 5, 2016, every other day, 4 doses were injected. At the same time, Zoladex was used as a combination treatment as recommended by the doctor.

4. Results:

In this example, subjects administered the compositions of the present disclosure did not show any obvious side effects except for muscle stiffness due to prolonged injection. During treatment, total prostate-specific antigen levels decreased, health conditions improved, and pain relieved. Prior to treatment, prostate perforation biopsy results showed that all 10 needles tested were positive, which number decreased to 5 out of 10 positive after treatment.

Example 15. Therapeutic effect of the composition on rectal cancer

1. Subject:

Sun, a 63-year-old male, was diagnosed with rectal cancer in November 2012 and underwent rectal cancer resection + liver biopsy. Postoperative pathology showed moderately differentiated rectal ulcerative carcinoma, infiltration of the entire intestinal wall, multiple cancer nodules visible inside mesenteric adipose tissue, visible metastatic cancer in lymph nodes, and metastatic nodules visible in the liver. After recovery from surgery, treatment was performed with bevacizumab (600 mg) and Xelox (OXA 250 mg, Xelod3.0, 14d) starting in May 2012. After 8 cycles, only bevacizumab was used until September 2015. A CT imaging diagnosis taken in March 2018 reported multiple intrahepatic metastases, multiple bony metastases throughout the body, and confirmed metastases to the left lower lobe nodule in the left lung.

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

With the patient's consent, YS-ON-001 was administered via intramuscular injection starting on March 25, 2016. During the first month, 1 dose (1 ml) was administered every other day. In month EN, 1 dose was administered daily. In the third month, 2 doses were administered daily. From August 10, 3 doses were administered daily.

4. Results:

In this example, subjects administered the compositions of the present disclosure did not show any apparent side effects. During treatment, the patient's symptoms did not change, and the disease was stable without further complications. The lower extremity edema induced by the use of bevacizumab was improved.

Example 16. Therapeutic effect of composition on endometrial cancer

1. Subject:

Zhang, a 56-year-old woman, was diagnosed with stage 3 endometrial cancer in December 2010 and underwent extensive abdominal hysterectomy + radioactive ovarian cancer surgery. The postoperative pathology was endometrial adenocarcinoma (histologic grade 1), bilateral ovarian cancer infiltration, and metastasis to the right uterine sacral ligament. After surgery, treatment with docetaxel + cisplatin chemotherapy was started in January 2011. In January 2015, liver and rectal recurrence and metastasis were found, followed by chemotherapy and cancer pain treatment.

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

With the patient's consent, YS-ON-001 was administered via intramuscular injection from June 10, 2016. 2 doses (2 ml) were administered daily for the first 10 days, followed by 4 doses (4 ml) daily.

4. Results:

In this example, subjects administered the compositions of the present disclosure did not show any apparent side effects except for muscle stiffness at the injection site. After 2 months of treatment, compared with CT examination of the previous month, there was a slight decrease in the low-density mass of the left abdominal wall, there was a clear irregular thickening in the proximal splenic curve, and it was slightly better than before.

Example 17. Therapeutic effect of the composition on ovarian cancer

1. Subject:

Zhou, a 56-year-old woman, was diagnosed with stage IIIc ovarian cancer (high-grade malignant carcinoma) in June 2016 and underwent open surgery for ovarian cancer cell degeneration + rectal cancer resection. The postoperative pathology represents high-grade malignant carcinoma, rectal lesions, matched with (left appendage, right ovary), cancerous tissue found in the appendix mammary gland and abdominal wall.

2. Composition:

When administered to human subjects, YS-ON-001 is formulated in sterile water for injection.

3. Dosage Prescription:

With the consent of the patient, YS-ON-001 was administered via intramuscular injection starting on June 16, 2016. 2 doses (2 ml) were administered daily for the first 7 days, followed by 4 doses (4 ml) daily. At the same time, docetaxel and carboplatin were used as combination therapy as recommended by the doctor.

4. Results:

In this example, subjects administered the compositions of the present disclosure did not show any apparent side effects. During treatment, the patient's symptoms remained unchanged and were stable without further progression.

references

Any listing or discussion of documents expressly previously published herein is not necessarily to be taken as an admission that such documents are part of the state of the art or common general knowledge.

The following references may be of interest:

Wanqing Chen, et al. Cancer Statistics in China, 2015. Cancer Journal for Clinicians. 2016 Vol. 66:1 15-132.

Wanqing Chen, et al. Cancer Statistics in China, 2015. Cancer Journal for Clinicians. 2016 Vol. 66:1 15-132.

Guoqian Kuang, et al. Current Status and Prospect of Cancer Virotherapy Clinical Studies. Journal of Guangxi Medical University, 1995 Vol. 12:617- 619.

Guoqian Kuang, et al. Current Status and Prospect of Cancer Virotherapy Clinical Studies. Journal of Guangxi Medical University, 1995 Vol. 12:617-619.

Shounan Tan, Fengyu Zhang. Studies on Rabies and Rabies Vaccine for Human Use. Medical Information 201 1 Vol. 24:2841 -2842.

Shounan Tan, Fengyu Zhang. Studies on Rabies and Rabies Vaccine for Human Use. Medical Information 201 1 Vol. 24:2841 -2842.

Yuhui Zhang. The Establishment of Rabies Vaccine Purification Technology. Chinese Journal of Biologicals 1999 Vol. 12 Iss. No. 4:231 -232.

Yuhui Zhang. The Establishment of Rabies Vaccine Purification Technology. Chinese Journal of Biologicals 1999 Vol. 12 Iss. No. 4:231 -232.

Jiang Zhong. Oncolytic Virus and Tumor Treatment, Foreign Medicine (Microbiology Section), 2004 Vol. 27 Iss. No. 6.

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Kenney S, et al. Viruses as oncolytic agents: a new age for therapeutic virus. J Nati Cancer Inst, 1994,86:1185.

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CN100341571C.

CN100341571C.

RU2414238C2.

RU2414238C2.

US2010/0297072A1.

US2010/0297072A1.

WO2009/016433.

WO2009/016433.

Claims (28)

A composition for use in the treatment of lung cancer, comprising:
a) polyinosinic acid-polycytidylic acid (PIC),
b) an antibiotic or polyamine compound;
c) a cation, and
d) contain viruses;
wherein the virus cannot replicate in a human subject and is inactivated. According to claim 1, wherein the virus is Ravdoviridae, Adheniviridae, Arenaviridae, Astroviridae, Buniaviridae, Cliciviridae, Flaviviridae, Hepatitis delta virus, Hepeviridae, Order Mononegavirus, Order Nidovirus. , Piconavirus family, Orthomyxovirus family, Papillomavirus family, Parvovirus family, Polyomavirus family, Poxvirus family, Reovirus family, Retroviridae and Togavirus family. Composition, characterized in that selected from the group consisting of. The composition according to claim 2, wherein the virus belongs to the genus lyssavirus of the family rhabdoviridae. The composition according to claim 3, wherein the virus is a rabies virus. According to claim 1, wherein the antibiotic is tacromycin, anthracycline, butyrine sulfate, gentamicin, hygromycin, amikacin, dideoxy kanamycin, nebramycin, β-lactam, neomycin, puromycin, A composition selected from the group consisting of streptomycin, streptozocin, and any combination thereof. The method of claim 1, wherein the polyamine compound is arginine salt, spermidine, N-(3-aminopropyl), N-(3-aminopropyl)-1,4-butanediamine, spermine, OS-dimethylaminothio A composition selected from the group consisting of phosphate, poly-lysine, aminoglycoside, and any combination thereof. The cation of claim 1 , wherein the cation is selected from the group consisting of calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, zinc, and any combination thereof. A composition comprising The method of claim 1 , wherein the ratio of the virus to the PIC is 1 IU/50 μg, 1 IU/60 μg, 1 IU/70 μg, 1 IU/80 μg, 1 IU/90 μg, 1 IU/100 μg, 1 IU/125 μg, 1 IU/200 μg, 1 IU/250 μg, 1 IU/300 μg, 1 IU/350 μg, 1 IU/400 μg, 1 IU/450 μg, 1 IU/500 μg, 1 IU/550 μg, 1 IU/600 μg, 1 IU/700 μg, 1 IU/800 μg, 1 IU/1OOO μg, 1 IU/1500 μg, 1 IU/2000 μg, 1 IU/2500 μg, 1 IU/3000 μg, 1 IU/ 4000 μg, 1 IU/5000 μg, 1 IU6000 μg, 1 IU/7000 μg, 1 IU/8000 μg, 1 IU/9000 μg, 1 IU/10000 μg, and in a range between any two of the above ratios. A composition selected from the group consisting of. The composition according to claim 1, wherein the amount of the PIC is 250 μg to 5000 μg per unit dose. 10. The composition of claim 9, wherein the amount of PIC is selected from the group consisting of 250 μg, 500 μg, 1000 μg, 1500 μg, 2000 μg, 3000 μg, 4000 μg and 5000 μg per unit dose. The composition of claim 1, wherein the amount of the virus is 0.1 IU to 100.0 IU per unit dose. The method of claim 11, wherein the amount of virus per unit dose is 0.5 IU, 1.0 IU, 1.5 IU, 2.0 IU, 2.5 IU, 3.0 IU, 3.5 IU, 4.0 IU, 5.0 IU, 6.0 IU, 7.0 IU, 8.0 IU, 9.0 IU, 10.0 IU, 15.0 IU, 20.0 IU, 30.0 IU, 40.0 IU, 50.0 IU, 60.0 IU, 70.0 IU, 80.0 IU, 90.0 IU, 100.0 IU, and a range between any two of the above amounts. A composition characterized in that it is selected from. The composition according to claim 11, wherein the amount of the virus is 0.5 IU to 10.0 IU per unit dose or 1.0 IU to 5.0 IU per unit dose. 14. The method according to any one of claims 9 to 13, wherein the unit dose is 0.1 ml, 0.15 ml, 0.2 ml, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, 4.0 ml, 5.0 ml, 10.0 ml, 20.0 ml, 30.0 ml, 40.0 ml, 50.0 ml, 60.0 ml, 70.0 ml, 80.0 ml, 90.0 ml, 100.0 ml, 150.0 ml, 200.0 ml, 250.0 ml, and between any two volumes A composition characterized in that it is prepared in a volume selected from the group consisting of: The composition according to claim 1, wherein the amount of the virus is 0.05 IU/ml to 40.0 IU/ml in concentration. 14. The composition of any one of claims 1 to 13, wherein the composition comprises gelatin, sucrose, sugar, lactose, maltose, trehalose, glucose, low molecular weight dextran, sorbitol, polysorbate 20, mannitol polyethylene glycol, Human serum albumin, recombinant albumin, sodium octanoate, urea, aluminum hydroxide, phenol red, magnesium chloride, potassium chloride, sodium chloride, sodium thiosulfate, potassium dihydrogen phosphate, ascorbic acid, trichloromethane, phenol, and thimerosal and at least one auxiliary substance selected from the group consisting of any combination thereof. 14. The method of any one of claims 1 to 13, wherein the composition comprises at least one physiologically selected from the group consisting of acetate, tris(hydroxymethyl)aminomethane, bicarbonate, carbonate, phosphate buffer and any combination thereof. Composition, characterized in that it further comprises an acceptable buffer. 14. The composition according to any one of claims 1 to 13, wherein the pH of the composition is between pH 6.5 and pH 8.0. The composition of claim 1 , wherein the composition is prepared in a solid dosage form or a liquid dosage form; said liquid dosage form is selected from the group consisting of injectable solutions, suspensions, ointments, emulsions, drops, syrups, and gels; wherein the solid dosage form is selected from the group consisting of dry powder or lyophilized powder, tablets, capsules, suppositories, granules, and dragees. 14. The composition according to any one of claims 1 to 13, wherein the cancer is not caused by a virus. 14. The composition of any one of claims 1 to 13, wherein the composition comprises intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, intradermal, intranasal, intraocular, oral, sublingual, intratumoral, and peritumoral. A composition, characterized in that it is administered by a route selected from the group. According to claim 1, wherein the composition is once a month, twice a month, 3 times a month, 4 times a month, 5 times a month, 6 times a month, 7 times a month, 8 times a month, once a week, twice a week, a week 3 times a week, 4 times a week, 5 times a week, 6 times a week, once every 3 days, twice every 3 days, 3 times every 3 days, once every 2 days, twice every 2 days, once a day and administered to the human subject at a frequency selected from the group consisting of twice per day. According to claim 1, wherein the composition is
A therapeutically effective amount is administered to a human subject via intramuscular injection at a frequency of twice every 3 days, or
A composition, wherein a therapeutically effective amount is administered to a human subject via intramuscular injection at a frequency of once a week. 14. The composition according to any one of claims 1 to 13, wherein the cancer is a metastatic tumor. 14. The composition according to any one of claims 1 to 13, wherein the composition is used in combination with an anti-tumor treatment regimen. 26. The method of claim 25, wherein the anti-tumor treatment regimen is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, and immunotherapy, wherein the therapeutic agent used in the chemotherapy is an alkylating agent, an anti-metabolic anti-neoplastic agent. , anti-tumor antibiotics, anti-tumor plants, platinum compound anti-neoplastic agents, hormone balancing anti-neoplastic agents, and several types of anti-neoplastic agents, wherein the therapeutic agent used in the targeted therapy is rituximab, selected from the group consisting of bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, and sorafenib, wherein the therapeutic agent used in the immunotherapy is a PD-1 inhibitor, a PD-L1 inhibitor and selected from the group consisting of CTLA4 inhibitors,
wherein said alkylating agent is selected from the group consisting of cyclophosphamide, ifosfamide and thiotepa, and said antimetabolic antineoplastic agent is selected from the group consisting of methotrexate, mercaptopurine, fluorouracil and cytarabine; wherein the anti-tumor antibiotic is selected from the group consisting of bleomycin, daunorubicin, actinomycin D, mitomycin, doxorubicin and mitoxantrone, and wherein the anti-tumor plant is vincristine, etoposide, teni wherein said platinum compound anti-neoplastic agent is selected from the group consisting of cisplatin, carboplatin and oxaliplatin, said hormone balancing anti-neoplastic agent is leuprolide, tamoxifen, flu A composition selected from the group consisting of tamide and formestane, wherein the various kinds of anti-neoplastic agents are arsenic trioxide. A kit for use in the treatment of lung cancer, comprising:
A kit comprising at least one container, wherein each container independently comprises a composition according to any one of claims 1 to 13 and 19. 28. The kit of claim 27, wherein the kit further comprises an item selected from the group consisting of a needle, water for injection, instructions for use, and any combination thereof.

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